Expression of recombinant human insulin-like growth factor I in mammalian cells.
In order to develop a suitable mammalian expression system for human insulin-like growth factors (hIGFs) and mutant IGFs, we have constructed several artificial IGF genes, based on a cDNA encoding the IGF-I precursor (153 amino acids). Transient expression experiments using mouse Ltk- cells revealed that the IGF-I gene constructs were efficiently expressed when placed under control of the SV40 Early promoter (SV40E). This resulted in the synthesis and secretion of IGF-I receptor-reactive products. Constructs encoding an IGF-I precursor with a truncated signal peptide of 25 amino acids under control of SV40E promoter or the inducible Drosophila heat shock hsp70 promoter, were used to establish stably transformed CHOdhfr- and mouse L cells. Clones secreting IGF-I were identified by an IGF-I-specific radioreceptor assay. Immunoblot analysis of conditioned media from these clones resulted in the specific precipitation of a protein of 7 kDa identical in size to native IGF-I purified from human serum. After optimization of the expression conditions, the stable cell lines secrete 0.5-2 microgram/10(6) cells of IGF-I. The biological activity of the secreted recombinant IGF-I was shown by its ability to stimulate DNA synthesis in human MCF-7 cells. The results described in this paper indicate that a mammalian expression system, employing CHOdhfr- or L cells, is a useful system for the synthesis of biological active IGF-I.
Bovenberg
W A
WA
Institute of Molecular Biology and Medical Biotechnology, State University of Utrecht, The Netherlands.
Dauwerse
J G
JG
Pospiech
H M
HM
Van Buul-Offers
S C
SC
Van den Brande
J L
JL
Sussenbach
J S
JS
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D007739:L Cells; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I